Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
  • A61B5/00—Measuring for diagnostic purposes; Identification of persons
  • A61B5/0059—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence
  • A61B5/0082—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence adapted for particular medical purposes
  • A61B5/0084—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence adapted for particular medical purposes for introduction into the body, e.g. by catheters
  • A61B5/0086—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence adapted for particular medical purposes for introduction into the body, e.g. by catheters using infrared radiation
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
  • A61B5/00—Measuring for diagnostic purposes; Identification of persons
  • A61B5/0059—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence
  • A61B5/0071—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence by measuring fluorescence emission
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
  • A61B5/00—Measuring for diagnostic purposes; Identification of persons
  • A61B5/0059—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence
  • A61B5/0075—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence by spectroscopy, i.e. measuring spectra, e.g. Raman spectroscopy, infrared absorption spectroscopy
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
  • A61B5/00—Measuring for diagnostic purposes; Identification of persons
  • A61B5/72—Signal processing specially adapted for physiological signals or for diagnostic purposes
  • A61B5/7235—Details of waveform analysis
  • A61B5/7253—Details of waveform analysis characterised by using transforms
  • A61B5/7257—Details of waveform analysis characterised by using transforms using Fourier transforms
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/65—Raman scattering
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
  • A61B2562/00—Details of sensors; Constructional details of sensor housings or probes; Accessories for sensors
  • A61B2562/02—Details of sensors specially adapted for in-vivo measurements
  • A61B2562/0233—Special features of optical sensors or probes classified in A61B5/00
  • A61B2562/0242—Special features of optical sensors or probes classified in A61B5/00 for varying or adjusting the optical path length in the tissue
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
  • A61B5/00—Measuring for diagnostic purposes; Identification of persons
  • A61B5/72—Signal processing specially adapted for physiological signals or for diagnostic purposes
  • A61B5/7203—Signal processing specially adapted for physiological signals or for diagnostic purposes for noise prevention, reduction or removal
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/65—Raman scattering
  • G01N2021/653—Coherent methods [CARS]
  • G01N2021/656—Raman microprobe

Definitions

• Atherosclerotic vessels are at present furnished almost exclusively by angiography, which provides anatomical information regarding plaque size and shape as well the degree of vessel stenosis. The decision of whether an interventional procedure is necessary and the choice of appropriate treatment modality is usually based on this information.
• composition of atherosclerotic plaques vary
• Plaque biopsy is
• optical spectroscopic methods as a means of assessing plague deposits.
• Such "optical biopsies” are nondestructive, as they do not require removal of tissue, and can be performed rapidly with optical fibers and arterial catheters. With these methods, the clinician can obtain, with little additional risk to the patient, information that is necessary to predict which lesions may progress and to select the best treatment for a given lesion.
• optical methods most attention has centered on ultraviolet and/or visible fluorescence. Fluorescence spectroscopy has been utilized to diagnose disease in a number of human tissues, including arterial wall. In arterial wall,
• fluorescence of the tissue has provided for the characterization of normal and atherosclerotic artery.
• the information provided is limited by the broad line width of fluorescence emission signals.
• fluorescence based methods provide information about the electronic structure of the constituent
• Atherosclerosis as well as other diseases which affect the other organs of the body.
• the present invention relates to vibrational spectroscopic methods using Fourier transform infrared (FT-IR) attenuated total reflectance (ATR) and near-infrared (IR) FT-Raman spectroscopy. These methods provide extensive molecular level
• a preferred embodiment utilizes FT-Raman spectra of human artery for distinguishing normal and atherosclerotic tissue.
• Near IR FT-Raman spectroscopy can provide information about the tissue state which is unavailable from fluorescence methods.
• These methods include the steps of irradiating the tissue to be diagnosed with radiation in the infrared range of the electromagnetic spectrum, detecting light emitted by the tissue at the same frequency, or alternatively, within a range of frequencies on one or both sides of the irradiating light, and analyzing the detected light to diagnose its condition.
• Both the Raman and ATR methods are based on the acquisition of information about molecular vibrations which occur in the range of wavelengths between 3 and 300 microns. Note that with respect to the use of Raman shifted light, excitation wavelengths in the ultraviolet, visible and infrared ranges can all produce diagnostically useful information.
• Near IR FT-Raman spectroscopy is ideally suited to the study of human tissue.
• Raman spectroscopy is an important method in the study of biological samples, in general because of the ability of this method to obtain vibrational spectroscopic information from any sample state (gas, liquid or solid) and the weak interference from the water Raman signal in the "fingerprint" spectral region.
• the FT-spectrometer furnishes high throughput and wavelength accuracy which might be needed to obtain signals from tissue and measure small frequency shifts that are taking place.
• Standard quartz optical fibers can be used to excite and collect signals remotely.
• Near IR FT-Raman spectroscopy provides the capability to probe biological substituents many hundred microns below the tissue surface. In particular, for atherosclerotic tissue, calcified deposits below the tissue surface are easily
• the ATR technique offers several features especially suited to sampling of human tissue
• the ATR method can non-destructively probe internal human tissue either by direct contact in a hollow organ (e.g. artery), or by insertion of a needle probe.
• a hollow organ e.g. artery
• a needle probe e.g. a needle probe.
• strong water absorption dominates the spectra of highly hydrated samples such as arterial tissue, obscuring the absorption from other tissue components (see Figure 8).
• Accurate subtraction of the strong water absorption from FT-IR ATR spectra is relatively easy and very reliable with the high dynamic range, linearity, stability, and wavelength precision of available FT spectrometers.
• mid-IR spectra of aqueous protein solutions can be collected with fiber optic ATR probes.
• Such probes are easily adaptable to existing catheters for remote, non-destructive measurements in vivo.
• the mid-IR ATR technique allows clinicians to gather precise histological and biochemical data from a variety of tissues during standard catheterization procedures with minimal additional risk.
• the present methods relate to infrared methods of spectroscopy of various types of tissue and disease including cancerous and pre-cancerous tissue, non-malignant tumors or lesions and
• Another preferred embodiment of the present invention uses two or more diagnostic procedures either simultaneously or sequentially collected to provide for a more complete diagnosis. These methods can include the use of fluorescence of endogenous tissue, Raman shifted measurements and/or ATR measurements.
• Yet another preferred embodiment of the present invention features a single stage spectrograph and charge-coupled device (CCD) detector to collect NIR Raman spectra of the human artery.
• CCD charge-coupled device
• Raman spectra can be collected by the CCD at two slightly different illumination
• Figures 1A-1C are schematic illustrations of preferred systems for providing the spectroscopic measurements of the invention.
• Figure 2 graphically illustrates FT-Raman spectra of human aorta: a) normal artery;
• Figure 3 graphically illustrates FT-Raman spectra of normal human aorta: a) irradiated from intimal side (spectrum multiplied by 3); and b) irradiated from adventitial side (primary adipose tissue). c) NIR FT-Raman spectrum of triglyceride, triolein.
• Figure 4 graphically illustrates FT-Raman spectra from human aorta: a) fibrous plaque; and b) atheromatous plaque, c) FT-Raman spectrum of cholesterol monohydrate powder.
• Figure 5 graphically illustrates FT-Raman spectra of calcified human aorta: a) calcified with fibrous cap; b) excised calcification from a
• Figure 6 graphically illustrates FT-Raman spectra of calcified human aorta: a) calcified plaque with a fibrous cap (spectrum multiplied by 8); and b) exposed calcification.
• Figure 7 graphically illustrates the measured NIR Raman intensity of the 960 cm -1 band (A(960 cm -1 ) indicates the area of this band) in a calcified deposit as a function of depth below the irradiated surface.
• the dashed curve corresponds to the fit of an exponential function to the data with an exponent of 2.94 mm -1 .
• Figure 8 graphically illustrates FT-IR ATR spectra (4000 - 700 cm -1 ) of (a) normal aorta, intimal surface; and (b) buffered saline (0.14M NaCl,pH 7.4).
• Figure 9 graphically illustrates FT-IR ATR spectra (1800 - 800 cm -1 ) after water subtraction: (a) Normal aorta, intimal surface; (b) Sub-adventitial fat; (c) Saline rinsed from the intimal surface of normal aorta.
• Figure 10 graphically illustrates FT-IR ATR spectra (1800 - 800 cm -1 ): (a) Two consecutive water-subtracted spectra of normal aorta, intimal surface, collected immediately after placement on ATR element (solid line) and 10 minutes later (dashed line); (b) Same two spectra as in (a) after lipid subtraction, scaled to have equal maxima.
• Figure 11 graphically illustrates FT-IR ATR spectra (1800 - 800 cm -1 ) , water-and lipid-subtracted: (a) Normal aorta, media layer; (b) Atherosclerotic plaque, intimal surface; (c)
• Atheromatous plaque with intact fibrous cap intimal surface.
• Figure 12 graphically illustrates FT-IR ATR spectra (1800 - 800 cm -1 ) : (a) Necrotic core of atheromatous plaque, water-and lipid-subtracted; (b) Dry film of cholesterol.
• Figure 13 graphically illustrates scatter plot for all samples of the area, A(1050), of the 1050 cm -1 cholesterol band (integrated from 1075 to 1000 cm -1 ) ratioed to the area, A(1550) of the
• FIBROUS includes atherosclerotic and atheromatous plaques with intact fibrous caps
• NECROTIC includes exposed necrotic atheroma cores
• Figure 14 graphically illustrates FT-IR ATR spectra (1800 - 800 cm -1 ): (a) Second derivative spectrum of normal aorta intima ( Figure 8a); (b) Water-subtracted spectrum of same normal aorta intima specimen (same as Figure 9a).
• Figure 15 graphically illustrates a scatter diagram for all the specimens of the area, A(1050) of the 1050 cm -1 cholesterol band plotted versus the area, A(1382), of the 1382 cm -1 cholesterol band. Both cholesterol bands have been normalized to the area, A(1050), of the protein amide II band. All band intensities were calculated from the water-and lipid-subtracted spectra. Tissue categories are the same as in Figure 13. The solid line represents a linear least squares fit to the data.
• Figures 16A and 16B are additional preferred embodiments of ATR probes adapted to make the diagnostic measurements of the present invention.
• Figure 17 is a schematic diagram of the system of Figure 1A modified to use the spectrograph/CCD Raman detector of the present invention.
• Figure 18 is a schematic diagram of a preferred system for implementing the spectrograph/CCD Raman detector of the present invention.
• spectrograph/CCD-Raman spectra of normal human aorta A) Raman plus fluorescence spectrum produced by illuminating the tissue sample with 810 nm laser light; B) Raman difference spectrum produced by subtracting spectra produced by illuminating the tissue sample with 810 and 812 nm laser light; C) resulting Raman spectrum produced by integrating the Raman difference spectrum of B).
• spectrograph/CCD-Raman spectra of an atherosclerotic plaque with a calcified deposit exposed at the surface A) Raman plus fluorescence spectrum produced by illuminating the tissue sample with 810 nm laser light; B) Raman difference spectrum produced by subtracting spectra produced by
• Figure 1A includes separate block diagrams for the system of the invention which utilizes laser light for
• the ablation laser 225, pulse stretcher 229 and the pulse filler/multiplexer 231, 233 produce an output laser ablation pulse of sufficient energy and intensity to remove tissue and sufficient pulse duration to propagate through a fiber optic laser catheter delivery system without damaging the fibers.
• a device 219 is used to contact the tissue such as multiple-fiber laser catheter 10 of Figure 1B with an optical shield.
• the catheter 10 is inserted into the artery and the distal end of the catheter is brought into contact with the lesion.
• a determination is made as to the type of tissue at which each optical fiber 20a-c' is aimed. Only fibers aimed at diseased tissue are activated. Thus, selective tissue removal is obtained.
• this technique is also applicable for guiding surgical procedures in other organs and tissue types such as the colon and bladder.
• the present invention relates to systems and methods of performing spectral diagnostics to diagnose the tissue in front of each fiber.
• a preferred embodiment a laser light source 207 that is coupled to the fibers.
• the diagnostic light is sent to the fiber of choice by the optical fiber selector 217.
• the diagnostic light exits the selected optical fiber and falls on the tissue.
• the tissue absorbs the light and a fraction of the absorbed light is re-emitted, by Rayleigh fluorescence, Raman or other elastic or inelastic light scattering processes.
• This light is returned to the optical fibers and exits from selector 217, and is detected by a photodiode, photomultiplier or other detector 203. Returning light could use different optical fibers than those employed for illumination. Diagnostic subsystem produces the entire spectral signal which is coupled to computer 80.
• the computer stores the information in a memory as a spectrum, which is a graph of light intensity vs. wavelength. This can be displayed immediately on the video display 82 or compared to an existing spectrum stored in the computer and the difference displayed on the spectral display 86. Temporal display 88 can display corrections made for the wavelength dependent sensitivities of the source. Information from either the temporal or spectral display can be stored in the computer 80. The comparative data is shown on numerical display 84 to provide a quantitative measure of the health of the tissue observed.
• a spectral or numerical display which indicates the type of tissue at which the fiber of interest is aimed. If the tissue is plaque, and is to be removed, then fiber selector 217 will align this fiber with the output beam of the high power laser 225. Then, the high power laser 225 is turned on and an appropriate power level is selected for a predetermined amount of time to remove a certain amount of diseased tissue. The beam of laser 225 is transmitted to pulse stretcher 229 and pulse filler/multiplexer 231, 233 to
• the procedure is repeated for different fibers. Where diseased tissue is detected, it is quickly removed.
• the laser catheter 10 nibbles away at the plaque, leaving the healthy artery wall intact.
• the laser catheter 10 will tend to make contact with artery wall 32 at the outside wall of the bend.
• the optical fiber 20c is not fired. The lesion is removed asymmetrically. This allows the laser catheter 10 to follow the lumen 39, 39a around the bend.
• the artery wall 32 is not irradiated and is not perforated. Additional details of this fiber optic catheter 10 are
• the methods can utilize Fourier transform detection to observe the radiation thereby providing improved signal/noise ratios.
• Other techniques e.g. diode array detection and CCD detection
• diode array detection and CCD detection can also be used.
• contributions from major tissue constituents can be "subtracted out” to reveal information about molecules which are present in small concentrations. For example, in ATR water contributions are removed before the "dry" tissue constituents could be studied. Also,
• ATR is "naturally" suited to probe surface disease, such as the superficial cancers of the bladder and GI tract, whereas Raman is well suited to providing information about conditions deep inside tissue (such as breast cancer or stones).
• the ATR tissue sampling depth can be controlled by properly matching the probe surface material to the tissue type.
• the ATR signal is very sensitive to the surface of the waveguide or probe. For example, if the probe surface has an affinity for lipids in the tissue, lipids can migrate to the probe surface, creating a thin lipid layer and producing a large signal. This can be a problem (it can give
• Probes with special surfaces can be developed to prevent this effect or to promote it, in order to search for particular substances in the tissue.
• Raman diagnostic methods permit adjustment of Raman depth by varying the wavelength.
• Raman sampling depth can be controlled to a large extent by probe design.
• Depth information is important if one desires to provide imaging by creating 3D images of small tumors in the brain or breast. Differential techniques based on the ideas of the preceding paragraph might allow accurate localization of such tumors in three dimensions.
• Near-IR Raman can be combined with a sound wave technique (time of flight or standing waves set up in the tissue) ⁇ the sound wave would modulate the Raman signal emanating from a point in the tissue when it passes that point, and the modulated signal could be used to establish the depth of the tissue producing the signal.
• a system employed for the collection of Raman spectral data from excised tissue samples is
• FT-Raman spectra were measured from 0 - 4000cm -1 below the laser excitation frequency with a FT-IR interferometer 40 equipped with a FT-Raman accessory.
• the accessory employed at 180 back scattering geometry and a cooled (77K) InGaAs detector 42.
• a 1064 nm CW Nd:YAG laser 44 can be used for irradiating a sample 46: utilizing 500 mW to 1 W laser power in a 1.0 to 2.5 mm spot 48 at the sample 46 to collect Raman data.
• a pulsed laser source can also be employed.
• sample 46 generated a beam 46 that is directed through plasma filter 48, mirrors 50, 52, focussing lens 54 and mirror or prism 56 before irradiating the sample 46.
• the radiation received by sample 46 undergoes various mechanisms of absorption, reflection and scattering including Raman scattering.
• Some of the light emitted by the tissue is directed toward lens 60 and then through one or more Rayleigh filters 62.
• the collecting lens 60 collects this backscattered light 64 and collimates it.
• the Rayleigh filters 62 removes the elastically scattered light and
• the tissue samples were placed in a suprasil quartz cuvette with a small amount of isotonic saline to keep the tissue moist, with one surface in contact with the irradiated by the laser 44.
• the spectra shown in Figures 2 through 6 were collected with 512 scans at 8 cm -1 resolution (approximately 35 minutes total collection time).
• Human aorta is composed of three distinct layers: intima, media, and adventitia.
• the intima normally less than 300 ⁇ m thick, is the innermost layer and provides a non-thrombogenic surface for blood flow. It is mainly composed of collagen fibers and ground substance.
• the medial layer typically about 500 ⁇ m thick, is quite elastic and serves to smooth the pulsatile blood flow from the heart.
• the structural protein elastin is the major component of aortic media, with some smooth muscle cells present as well.
• the outermost adventitial layer serves as a connective tissue network which loosely anchors the vessel in place, and is mainly made up of lipids, lipoproteins and collagen.
• the intima thickens due to collagen proliferation, fatty necrotic deposits accumulate under and within the collagenous intima, and eventually, calcium builds up, leading to calcium hydroxyapatite deposits in the artery wall.
• Figure 2a shows the FT-Raman spectrum of a full thickness section of aorta grossly identified as normal. Laser irradiation was on the intimal side. The dominant bands appear at 1669 cm -1 and 1452 cm -1 and can be assigned to an amide I backbone and C-H in-plane bending vibration from proteins,
• FIG. 3 Another example of a typical NIR FT Raman spectrum from normal aorta is shown in Figure 3.
• Figure 3a the major vibrational bands observed in normal aorta are all attributable to protein vibrations: the band at 1658 cm -1 is assigned to the amide I
• the spectrum of normal aorta is at least 25% weaker than any of the pathologic samples.
• the peak frequency of the C-H bending band, which averaged for all the normal specimens is 1451+1 cm -1 , is specific to the protein C-H bending mode (See below).
• the weak band near 1335 cm -1 which appears as a shoulder in many of the normal specimens, appears to be specific to elastin, and the weak band at 1004 cm -1 is likely due to phenylalanine residues.
• Figure 3b displays the NIR FT Raman spectrum of the adventitial side of normal aorta.
• the irradiated adventitial surface consisted of several millimeters of visible adipose tissue.
• the bands observed in this adipose material appear to be mainly due to lipid, and in particular triglyceride, with almost no contribution from protein. This is not unexpected, as the triglyceride content of adipose tissue is on the order of 60%.
• This band is distinguished from amide I by its peak frequency and its width, which in this case is 17 cm -1 FWHM.
• Amide I in contrast, is roughly 60 cm -1 wide.
• the dominant C-H bending band is shifted to 1440 cm -1 , characteristic of lipids. This band is about 3 times more intense in adipose tissue than in normal intima, probably a result of the greater number of C-H groups per unit volume in
• the frequencies and structures of the C-H bending and C-C stretching bands suggest that most of the fatty acid chains are in the gauche conformation.
• the FT-Raman spectrum obtained from a diseased artery, an atheromatous plaque, with a thick fibrous connective tissue cap and an underlying necrotic core is shown in Figure 2b.
• the necrotic core of an atheromatous plaque contains cellular debris as well as large accumulations of oxidized lipids and cholesterol.
• the band in the amide I region, peaking at 1665 cm -1 is distinctly narrower in this spectrum compared to normal aorta.
• the in-plane C-H bend, at 1444 cm -1 is relatively more intense and has a distinct shoulder at higher frequency.
• the intense C-H bending band occurs at 1440 cm -1 , characteristic of lipid material. This band is roughly twice as intense as the C-H bending band in normal aorta. The complete absence of a band at 1746 cm -1 indicates that this lipid is not triglyceride. In fact, this lipid appears to be predominantly cholesterols, as identified by the sharp, characteristic band at 700 cm -1 and comparison to the cholesterol spectrum shown in Figure 4c. Again, this is not surprising, since cholesterols accumulate in high concentrations in atherosclerotic lesions. Several of the bands between 1000 and 500 cm -1 are assignable to
• vibrational modes of the sterol rings include the bands at 959, 882, 844, 805, 700, 605, and 546 cm -1 .
• the presence of fatty acid chains in the atheromatous plaque spectra is evidenced by bands at 1300/1262 cm -1 and 1130/1088 cm -1 , due to C-H bending and C-C stretching vibrations, respectively. These bands may contain contributions from cholesterol as well.
• the relative intensities of the fatty acid band at 1300 cm -1 and the sterol ring bands suggest a mixture of free cholesterol and cholesterol-fatty acid esters.
• the relative intensities of the 1130 cm -1 C-C stretching and the 700 cm -1 sterol bands indicate that most of the fatty acid chains are in the gauche conformation, consistent with the predominance of unsaturated fatty acid chains in the cholesterol esters in these plaques. It is
• the NIR FT Raman spectra of some of the fibrous plaques contained two unique bands, at 1519 and 1157 cm -1 . The intensities of these bands are highly correlated, which suggests that they are due to a single component. These bands, which have been previously observed in visibly-excited Raman spectra of atherosclerotic plaques, are assigned to
• the amount of carotenoid in these plaques is probably much smaller than the amounts of cholesterols or proteins, but may be strongly pre- resonance enhanced (14). The carotenoid bands are observed only in this subset of fibrous plaques.
• the NIR FT Raman spectrum of calcified plaque containing a subsurface calcified deposit and an overlying soft fibrous cap, exhibits an intense, sharp, new band at 960 cm -1 ( Figure 6a).
• This band specific to calcified tissue, is assigned to the symmetric stretching vibration of phosphate groups (15), which are present in high concentrations in the solid calcium salts.
• the weaker phosphate antisymmetric stretch is also present at 1072 cm -1 .
• a symmetric stretching vibration of carbonate groups may also contribute to this latter band.
• the phosphate vibrations are easily observed from subsurface deposits in the calcified plaques: the 960 cm -1 band can be observed from deposits up to 1.5 mm beneath a soft tissue cap with the current signal-to-noise level (See below).
• the calcified plaque also displays protein vibrations from the fibrous tissue cap. These include amide I at 1664 cm 1 and amide III near 1257 cm -1 .
• the C-H bending band at 1447 cm -1 suggests a mixture of protein and lipid, and the weak band at 699 cm -1 is likely due to cholesterol that is either in the fibrous cap, the calcified deposit, or both.
• the present methods provide an IR FT-Raman technique for differentiating various stages of atherosclerosis in human aorta. They demonstrate that molecular level information is available using these methods. This information is useful for following the pathogenesis of the disease and in guiding the treatment of different lesions.
• the near IR FT-Raman method with its relatively deep penetration depth, is able to obtain spectroscopic signals from below the tissue surface, yielding details about the atheromatous necrotic tissue and sub-surface calcifications. These signals can be utilized with an optical fiber based imaging system to determine the content and composition of
• hydroxyapatite can be easily detected below the tissue surface, we wished to determine the depth limit of detection using the NIR FT Raman technique.
• NIR FT Raman technique ten 200 ⁇ m sections of aortic media were cut and placed one at a time over a large calcified deposit (6 ⁇ 6 ⁇ 3 mm), and the FT Raman spectra of the 960 cm -1 band monitored as a function of depth below the surface.
• the signal from the calcified deposit was detectable until the deposit was greater than 1.6 mm below the irradiated surface. Even slightly deeper depths could be probed if the focus of the collection optics was moved into the tissue.
• the two dimensional resolution of the NIR FT Raman signal for material below the tissue surface was then tested by placing 1 mm of aortic media above another calcified deposit, and moving the tissue transversely in two dimensions through the laser beam and collection lens.
• the FT Raman signal was observed to drop-off rapidly as the beam and collection optics moved from the calcified deposit.
• the detected FT Raman signal closely followed the geometry of the calcified deposit below the surface, despite the significant scattering of the overlying layer of tissue. This result suggest that the Raman scattered light may be utilized for imaging objects below the tissue surface with minimal image blurring due to elastic scattering in the tissue.
• a second spectroscopic method is also used to obtain molecular vibration information, attenuated total reflective (ATR) of infrared light.
• Human aorta was chosen as an example to
• tissue components including collagen, elastin, and cholesterol to assist in analysis of the spectra.
• the ATR sampling crystal is a rod of high refractive index material which acts as a waveguide for the infrared sampling beam.
• This waveguide can be in the form of a needle that is adapted for penetration into the tissue to be diagnosed.
• the probe will have a geometry suitable for contacting the surface of exposed tissue sites or for contacting internal locations with a catheter.
• FIGs 16A and 16B illustrate preferred embodiments of the invention adapted for ATR diagnostic measurements within the human body.
• a single-ended probe 100 is shown where one or more optical fibers 102 both the incident light to, and the transmitted
• a 100% infrared reflector 106 such as gold is placed at the distal surface 108 of the ATR element 104 functions to return the transmitted light back through the same fiber as well as to provide double pass
• the ATR element 104 can be a separate component optically fastened to the optical fibers 102, or alternatively, it can be constructed from the end of the optical fiber by removing the
• Sampling is provided by placing the ATR element in contact with the tissue 110 of interest. Radiation is transmitted 112 and
• the probe can either be inserted through a standard endoscope or catheter to sample a hollow organ, or, if made with sufficiently thin optical fiber, it can be directly inserted directly into a solid organ as in the case of needle biopsy.
• the distal tip 108 is in the form of a needle.
• configuration on the end of the catheter can be long or shallow.
• a double-ended probe is illustrated in Figure 16B.
• Incident IR beam from FT-IR is transmitted through IR optical fiber 122 to ATR element 128 positioned at the distal end of catheter body 120.
• the ATR element is placed in contact with tissue 126 surface to be sampled. Transmitted light is
• the ATR element may be a
• the specimen to be sampled is placed in optical contact with the surface of the waveguide or ATR element.
• the evanescent wave which extends outside of the waveguide surface is absorbed by the sample in proportion to its absorption coefficient.
• the penetration depth of the evanescent wave into the sample depends on the wavelength of the infrared radiation and the refractive indices of the
• this depth is roughly 1 ⁇ m from 1800 to 700 cm -1 .
• n Z 2 sin 2 ⁇ -n w 2 )1 ⁇ 2 ⁇ /2 ⁇ (n Z 2 sin 2 ⁇ -n w 2 )1 ⁇ 2, where ⁇ is wavelength, ⁇ is angle of incidence and n z and n w are the refractive indices of ZnSe and water respectively.
• Figure 8 shows FT-IR ATR spectra of (a) normal aorta (intimal side) and (b) buffered saline. A comparison of these spectra shows that a majority of the IR absorption of normal intima can be attributed to water, which comprises roughly 80% of the tissue by weight.
• the large, broad bands peaking at 3300 cm -1 and 1636 cm -1 are due to the O-H stretching and H-O-H bending vibrations, respectively, of water, and the weak band at 2120 cm -1 is due to a water combination vibration.
• the 3300 cm -1 and 1636 cm -1 bands also include contributions from the N-H stretching and amide I vibrations.
• the relatively flat absorption between 1500 and 900 cm -1 and the rising absorption below 900 cm -1 is also due
• polypeptide backbone of repeating amide groups is the dominant element.
• repeating hydrocarbon chain is the defining quality. The end result is that these molecular units are present in very large concentrations, and their vibrational bands tend to dominate the spectrum.
• the lipid component observed in the tissue appears to be due to free lipid particles that have equilibrated with the tissue surface water, forming a thin water-lipid film on the tissue surface which is in full optical contact with the
• the ATR element immediately after the tissue specimen is placed upon the crystal.
• the tissue components beneath this film presumably achieve better optical contact with the ATR crystal as the sample settles.
• the content of lipid in a spectrum of aorta intima or media may be influenced by the presence of sub-adventitial fat in the specimen, and the relative lipid-protein absorbencies are accurate to 50% at best with the present experimental design. For the reason, all of the remaining spectra shown are both water and lipid subtracted.
• the major bands in the spectrum may be assigned to protein backbone vibrations. These include the bands at 1648 cm -1 (amide I), 1549 cm -1 (amide II), 1455 cm -1 (C-H bend), 1401 cm -1 (amide C-N stretch), and 1244 cm -1 (amide
• the protein C-H bending band at 1455 cm -1 is distinct from the corresponding vibration in lipid, which occurs as a double-peaked band at 1465/1457 cm -1 .
• FIG. 11a A typical spectrum of the medial layer of normal aorta is shown in Figure 11a. A comparison of this spectrum to that of normal intima ( Figure 10b) fails to reveal any significant differences.
• Atheromatous plaque are shown in Figures 11b and 11c, respectively.
• Figures 11b and 11c For these plaques, only the intact fibrous cap at the intimal surface is probed due to the short penetration depth (1 ⁇ m) of the beam. Any necrotic, atheromatous material beneath this fibrous cap is not sampled. Even so, the fibrous caps of these plaques are known to be compositionally different than normal intima and one might expect these differences to be reflected in the IR ATR spectrum. However, as in the case of media, no consistent differences are observed in the spectra of these plaques ( Figures 11b and 11c) and normal intima ( Figure 10b). This issue will
• Atheromatous core of an atheromatous plaque ( Figure 12a) as compared with the corresponding spectra of normal intima ( Figure 10b) as well as those of intact atherosclerotic ( Figure 11b) and atheromatous ( Figure 11c) plaques.
• the necrotic core was presumably exposed in vivo as disease progressed by ulceration of the overlying intimal fibrous tissue cap.
• the spectrum of necrotic core exposed by dissecting away the fibrous cap of a non-ulcerated atheromatous plaque is similar.
• a new band appears at 1050 cm -1 , with a secondary peak at 1023 cm -1 .
• necrotic core spectrum exhibits an increase and frequency shift in the 1466 cm -1 band as compared with the 1455 cm -1 protein band in normal intima as well as a set of unique bands near 1382 cm -1 .
• These characteristic bands are found in the spectra of all the exposed necrotic core samples and in none of the other samples (see below). The source of these unique bands in the
• necrotic core spectra may be cholesterol, which is known to accumulate in large amounts in atheromatous cores.
• An ATR spectrum of cholesterol (dry film) is shown in Figure 12b.
• the three major bands unique to the necrotic core, near 1463 cm -1 , 1382 cm -1 , and 1050 cm -1 match closely in position and relative intensities with the three main cholesterol bands at 1466 cm -1 , 1377 cm -1 , and 1056 cm -1 .
• Each of the main cholesterol bands has a secondary peak, which also appear to be present in the necrotic core bands.
• Atherosclerosis by mid-IR spectroscopy have been limited to date. It has been reported that ATR spectra have been recorded from partially dried human artery, among other tissues. In comparing a normal aorta from an infant to an atherosclerotic plaque in an adult, they observed increases in several bands in the atherosclerotic aorta. Most of these bands were associated with lipids and
• IR spectroscopy has been employed to determine the chemical composition of calcified atherosclerotic deposits.
• a more detailed IR study of atherosclerotic aorta involves recorded IR transmission spectra from thin layers sectioned at different depths into the arterial wall. Results showed increased absorption near 1739 cm -1 in the fatty (atheromatous) regions of plaque, which was attributed to absorption by cholesterol esters in the plaque. IR spectra from the fibrous tissue cap at the surface of the plaques were similar to normal intima.
• spectra collected with the ATR method are not equivalent to IR absorption spectra, but depend on properties of the ATR material and the sample in addition to the sample absorption coefficient. For instance, the penetration depth of the evanescent sampling wave depends on the refractive indices of the ATR material and the sample. However, the refractive indices of both ZnSe and human tissue are expected to vary slowly with frequency between 1800 and 700 cm -1 and such variations will at most affect the relative intensities of bands at different frequencies. All of the structure observed in the tissue spectra is attributed to absorption bands in the tissue.
• the component absorptions observed in an ATR spectrum also depends upon the optical contact of the sample and ATR element.
• the small penetration depth of the evanescent wave into the tissue sample implies that only a 5 ⁇ m thick layer, and preferably about 1 micron, of material at the surface is observed. This is referred to as the near surface region of the tissue for the purposes of this application.
• the tissue deeper than 5 microns from the surface is defined as the sub-surface region.
• This thin, sampled near-surface layer may differ in composition with the bulk sample. For example, a film of free water may be present on the surface of wet tissue, with different levels of some molecular species of the tissue relative to their
• the varied affinities for the ATR material of different moieties in the tissue may play an important role in the intensities of the observed bands.
• plaque fibrous cap intima and normal intima ATR spectra.
• ATR elements made of other substances with different biochemical affinities the spectral differences among these tissues can be substantially enhanced depending on the tissue type.
• the spectral lineshape of water varies rather slowly with frequency over much of the region of interest, especially between 1500 and 700 cm -1 . Therefore, any method which filters this slower variation and spares the sharper features of the non-water bands can separate the water and non-water components.
• the amide II band in normal intima ( Figure 14b) has a very weak shoulder near 1518 cm -1 , and the C-H bending region near 1468 cm -1 appears to include two overlapping peaks.
• the 1518 cm -1 band is clearly visible, and the C-H region exhibits two separate peaks at 1469 and 1456 cm -1 .
• the second derivative spectrum allows a more precise
• composition of tissue as determined from an ATR spectrum may not be precisely identical to the composition of the bulk tissue.
• the tissue composition can also be determined from overlapping bands by first deconvolving the bands of interest into their individual components. This is especially easy if one component has an additional, isolated band elsewhere in the spectrum.
• An example is the 1465 cm -1 C-H bending region, which is due to different tissue components with distinct spectral features in this region.
• this band is attributed to a combination of lipid and protein components. Since the lipid component also exhibits the isolated 1744 cm -1 band, this band can be used to subtract the lipid C-H bending component and isolate the protein C-H bending component at 1455 cm -1 ( Figure 10b), effectively deconvolving this band. Note that this deconvolution depends on having a reliable spectrum of one of the individual components, which, in this example, is the lipid spectrum in Figure 9b.
• the baseline-subtracted area of the 1050 cm -1 band, A(1050), is plotted versus that of the 1382 cm -1 band, A(1382), for all the samples,
• the present systems and methods demonstrate that infrared spectra of moist, bulk tissues can be reliably obtained with the ATR technique. Although water is the dominant absorber throughout much of the mid-infrared region, the high quality spectra acquired with the FT-IR ATR technique allow for accurate subtraction of the water signal.
• composition of arterial tissue non-destructively There methods are also applicable to the study and diagnosis of other tissues and tissue conditions, such as neoplasia.
• NIR Raman spectroscopy using a single stage spectrograph and a charge coupled device (CCD) detector offers superior sensitivity over the Nd:YAG excited FT-Raman system of Figures 1A and 1C.
• CCD charge coupled device
• a CCD can be used to detect the Raman scattered signals while still avoiding fluorescence excitation in most molecules.
• the system can operate usefully in the range of 750 nm to 1050 nm. Although the fluorescence emission from tissue is significantly higher with 810 nm than with 1064 nm excitation, the Raman signals are readily observed.
• the dominant noise source in the spectrograph/CCD system is shot noise associated with the fluorescence emission, which is 2-3 orders of magnitude smaller than the dark current noise of the InGaAs detector, which is the dominant noise source in the FT-Raman system.
• Figure 17 shows the laser diagnosis
• the diagnostic subsystem 201' includes a single stage spectrograph 310 and charge-coupled device (CCD) detector 312 for collecting near-infrared (NIR) Raman spectra from intact human arterial tissue.
• CCD charge-coupled device
• the fluorescence emission from human artery tissue is sufficiently weak to observe Raman bands more rapidly with the spectrograph/CCD system than with the 1064 nm excited FT-Raman system of Figures 1A and 1C.
• NIR Raman spectral data from excised tissue samples using a spectrograph and a charge coupled device (CCD) array is illustrated in Figure 18.
• NIR Raman spectra were measured from 100 - 2000 cm -1 below the laser excitation frequency with a single stage imaging spectrograph 310 (Acton Model ARC275, 0.25 m, F/3.8) and a CCD array 312
• System 300 can use a NIR 810 nm Nd:YAG pumped pulsed dye laser 314 operating at 10 Hz for
• a CW or diode laser source can also be employed.
• Laser 314 generated a laser beam 316 which is directed by mirror 318 through focusing optics 320 to impinge on sample 46 mounted behind a transparent window 321.
• the laser beam was focused on the sample at a 70° angle of incidence, yielding a spot size of 0.7 ⁇ 2 mm on the tissue surface.
• the average incident power at the sample was maintained at 20 mW to avoid excessive peak intensities during an individual pulse.
• the spectral signals were observed to be linear over a range of average incident powers from 2 to 20 mW.
• a portion of the scattered light 322 emitted by sample 46 was collected by collecting optics 324 at a 90° angle relative to the incident laser beam.
• Collecting optics 324 collimates and F/matches the collected light for the spectrograph 310.
• the collected light Prior to entering the entrance slit of the spectrograph 310, the collected light was passed through a series of Schott glass filters 326 which attenuated the elastically scattered component of the collected light.
• the combined effect of the Schott glass filters provided an optical density of 7 at 810 nm, a transmission of 20% at 850 nm (580 cm -1 from 810 nm), and a transmission of 85% above 900 nm (1200 cm -1 ).
• the spectrograph 310 utilized a 200 ⁇ m slit width and a 600 groove/mm grating blazed at 1 ⁇ m and could be scanned to provide spectral coverage over different wavelength regions.
• the 200 ⁇ m slit width provided a resolution of roughly 15 cm -1 .
• the CCD array 312 consisted of 298 (column) by 1152 (row) pixel elements having a total active area of 6.7 mm ⁇ 26 mm, with the short axis parallel to the slit.
• the CCD array was cooled to -110°C to eliminate dark current.
• Each row of pixels was binned to reduce readout noise.
• Commercially available CCD detectors offer extremely low detector noise and usable quantum efficiencies out to 1050 nm and provide substantial advantages over InGaAs and other NIR detectors. These advantages outweigh the lower throughput of the grating spectrograph, provided that broadband fluorescence interference is not too great with the shorter excitation
• Excised human aorta samples 46 obtained at the time of post-mortem examination were rinsed with isotonic saline solution (buffered at pH 7.4), snap-frozen in liquid nitrogen, and stored at -85°C.
• Atherosclerotic areas of tissue were identified by gross inspection, separated, and sliced into roughly 8 ⁇ 8 mm pieces.
• the tissue samples 46 were placed in a suprasil quartz cuvette with a small amount of isotonic saline to keep the specimens moist, and with one surface in contact with the transparent window 321 and irradiated by the laser 314.
• Raman spectra were typically measured between 100 cm -1 and 2000 cm -1 below the laser excitation frequency. Each spectrum was background subtracted to remove the DC offset of the A/D converter of the CCD controller. In addition, hot pixels due to high energy radiation events were removed from the recorded spectrum by applying a median filter having a 7 pixel wide window as to each spectrum. Raman frequencies were calibrated with the spectra of benzene and barium sulfate powder and are accurate to ⁇ 5 cm -1 . The spectra were not corrected for the wavelength dependent response of the filters, spectrograph, and CCD. For each spectrum shown in the following Figures, Raman signals were
• Figure 19A shows the Raman spectra of a normal aorta sample excited with 810 nm laser light and collected with the spectrograph/CCD system 300.
• the broadband background emission which is presumably due to tissue fluorescence, is roughly five times more intense than the strongest Raman bands at 1650, 1451, 1330, and 1253 cm -1 .
• the shot noise associated with detecting this background emission is substantially smaller than the Raman signals, allowing the Raman bands to be made distinct after the background emission signals are removed through filtering or subtraction.
• the shot noise is typically random noise exhibiting a Poisson distribution and is associated with the detector and/or the background emission itself.
• the fluorescence background emission from the arterial pathology tissue types described is 3 to 4 orders of magnitude larger than the Raman signals, and the shot noise associated with this stronger background emission completely obscures the Raman bands even after the background emissions are removed.
• the signal-to-noise ratio of the spectrum of normal aorta collected with the spectrograph/CCD system 300 with 20 mW incident power and 5 minutes collection time (Figure 19A) is similar to that obtained with the FT-Raman system of Figure 1C with 500 mW incident power and 35 minute collection time. Since the observed spectral signal-to-noise ratios are similar, we estimate that the noise level observed with the CCD detector 312 of Figure 18 is roughly 3400 times less than that observed with the InGaAs detector 42 of Figure 1C.
• the major noise source is the shot noise of the dark current
• the dominant noise source is the shot noise of the broadband tissue emission, as the dark current and readout electrons of the CCD are much smaller than this emission.
• the FT-Raman and spectrograph/CCD systems can be compared as follows.
• the incident intensity is 640 mW/mm 2 .
• the quantum efficiency of the InGaAs detector at 1200 nm is 0.7, and the FT-spectrometer throughput is 1.1 mm 2 sr, and the transmission efficiency of the FT-spectrometer and filters is roughly 0.062.
• the incident intensity is 14 mW/mm 2 .
• the CCD quantum efficiency is 0.15 at 900 nm
• the spectrograph throughput is 0.043 mm 2 sr
• the transmission efficiency of the spectrograph and filters is 0.24. Combining these factors and taking into account the v 4 dependence of the Raman cross-sections, the signal level measured by the FT-Raman spectrum is estimated to be 3400 times greater than that of the spectrograph/CCD spectrum.
• the collection time could be reduced by a factor of 40, to 8 seconds, with no change in the spectral signal-to-noise ratio.
• the noise level can be further reduced by using longer excitation wavelengths which minimize the tissue fluorescence emission.
• the optimum excitation wavelength also depends on the fluorescence excitation profile of the tissue. For tissue types that exhibit little fluorescence emission at visible wavelengths, such as colon and bladder tissue, the CCD can be operated at visible or near visible wavelengths to take advantage of increased quantum efficiency of the CCD at these wavelengths.
• the throughput of a 500 ⁇ m core, 0.2 numerical aperture fused silica optical fiber is 0.03 mm 2 sr, which is roughly the same as that of the spectrograph/CCD system. This means that the present lens collection system can be replaced with an optical fiber probe, as is required for in vivo operation, with no additional loss in signal.
• Figure 19A shows that although the shot noise due to the broadband tissue emission is relatively small, the sloping broadband fluorescence emission still obscures the sharper Raman signals and
• Figure 19B This operation is mathematically analogous to taking the derivative of the Raman spectrum, so that the original Raman spectrum can be recovered by integrating the difference spectrum, as shown in Figure 19C.
• the fluorescence background is greatly reduced in Figure 19C as compared with
• Some weaker bands may also be identified, such as the phosphate/carbonate band at 1070 cm -1 , although these are obscured by the large fluorescence
• the Raman spectrum of adventitial adipose tissue is shown in Figure 21, which can be compared to the FT-Raman spectrum shown in Figure 5c.
• the broadband emission is similar to that of normal aorta, while the Raman bands, due mainly to triglycerides in the tissue, are very strong, resulting in an excellent spectral signal-to-noise ratio.
• the spectrograph/CCD system with 810 nm excitation offers a faster alternative to FT-Raman with 1064 nm excitation and which has greater sensitivity.

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